Ferritin 2 for the host immunization against ticks

ABSTRACT

Ferritin 2 is a tick secreted protein, which serves in the tick plasma as a non-heme iron transporter from the gut to the peripheral tissues. It can be exploited as an antigen for raising up host antibodies, which can reversely block this antigen in the tick and the attached tick will not have functional mechanism of the non-heme iron transport from the gut to the peripheral tissues. This mechanism is essential for the further tick development and its blocking eventually prevents transmission of tick-borne pathogens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a national-stage application, claiming priority from International Application No. PCT/CZ2009/000085, filed Jun. 18, 2009, which application, in turn, claims priority from Czech Republic Application No. PV 2008-402, filed Jun. 25, 2008.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a protein antigen for immunization of the organism—the produced antibody will block an iron transport in the tick body and hereby prevent its long-lasting feeding on the host, eventually block transmission of pathogens from the tick to the host.

Until now, only intracellular ferritin 1 has been known in ticks, which was isolated, cloned and sequenced in our laboratory for the first time from two tick species—Ixodes ricinus and Ornithodoros moubata. Later on, other orthologs of ferritin 1 were sequenced and partially characterized from other tick species in different laboratories. Tick ferritin 1 is post-trascriptionally regulated by the IRP (iron-regulatory protein), which binds IRE (iron-responsive element) loop of the particular mRNA. Ferritin 1 is an oligomer (composed of 24 subunits) and functions in the intracellular iron storage and prevention of the undesirable oxidative effects caused by the free iron.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a list of the amino acid sequences of Ferritin 2 disclosed herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Ferritin 2 is a newly identified and characterized protein, which is secreted into the tick plasma. It has been identified in ticks Ixodes ricinus, Ixodes scapularis, Dermacentor variabilis, and Rhipicephalus microplus (formerly Boophilus microplus). We have sequenced these ferritins and determined their primary structures. Ferritin 2 functions in the tick body as a transporter of iron, which is obtained from the host blood (plasma). When we succeed to block the protein, the attached tick will not be able to feed on the host, because the mechanism of non-heme iron transport from the gut to the peripheral tissue will not be functional, which is essential for tick parasitic life. The blocking of ferritin 2 can be achieved by the reaction with the specific antibody, what leads to the formation of immunocomplex. Specific antibodies will be raised up in the host organism after immunization with the tick antigen ferritin 2. A recombinant ferritin 2 or its fragment expressed in the prokaryotic (Escherichia coli, etc.), or eukaryotic (Pichia pastoris, insect cells, etc.) expression systems can be used as a suitable antigen for the immunization. Expressed and purified recombinant ferritin 2 could be used for immunization with the appropriate adjuvants or formulation media. The advantage of use of ferritin 2 is its substantial amino-acid sequence difference from the mammalian heavy (H) or light (L) chain ferritins. This substantially reduces a risk of undesirable side effects of the vaccine after the host immunization.

Ticks with decreased synthesis of ferritin 2, caused by RNA interference, are not able to finish their feeding and usually dry and die directly on the host. This fact makes ferritin 2 an ideal candidate for development of vaccine or other compound that will interfere with ferritin 2. Disruption of the tick feeding on the immunized host can impair the normal tick development and possibly will lead to the elimination of pathogen transmission by ticks (e.g., Lyme disease spirochetes), which are dependent on the successful tick feeding.

Examples Example 1

Ferritin 2, designated as FER2-IR, is a protein produced by the tick Ixodes ricinus and its primary structure is determined by the amino-acid sequence:

(SEQ ID. NO: 1) MKQFVVILALIGAATSGNNLFENLDKYPLQDECQAALQEHINVEMHAS LVYMQMAAHFDNNKVARKGFSTFFAENSKEEREHAQKIIDYINKRGST VSLVNIDMPLITTWKSVLQALRDAISLENKVTNKLHAVHKIADEECKD PQLMDFIESEFLEEQVNSIDKLQRMITVLSNMDSGTGEYLLDRELLGD KKEF.

The signal sequence responsible for the protein secretion of the cell is marked in bold. The primary sequence enables correct folding of the protein to the quarter structure, which transports the non-heme iron from the tick gut to the peripheral tissues. In the absence of ferritin 2, or in the case of its successful blocking, the ticks are not able to finish feeding and dry directly on the host.

Example 2

Ferritin 2, designated as FER2-IS, is a protein produced by the tick Ixodes scapularis and its primary structure is determined by the amino-acid sequence.

(SEQ ID. NO: 2) MKQFVVLLALIGAATSGNNLFENLDKYPLQDECQAALQEHINVEMHA SLVYMQMAAHFDNNKVARKGFSTFFAENSKEEREHAQKIIDYINKRG STVSLVNIDMPQITTWKSVLQALRDAISLENKVTNKLHAVHKTADEE CKDPQLMDFIESEFLEEQVTSIDKLQRMITVLSNMDSGTGEYLLDRE LLGDKKEF.

The signal sequence responsible for the protein secretion of the cell is marked in bold. The primary sequence enables correct folding of the protein to the quarter structure, which transports the non-heme iron from the tick gut to the peripheral tissues. In the absence of ferritin 2, or in the case of its successful blocking, the ticks are not able to finish feeding and dry directly on the host.

Example 3

Ferritin 2, designated as FER2-RM, is a protein produced by the tick Rhipicephalus microplus and its primary structure is determined by the amino-acid sequence:

(SEQ ID. NO: 3) MLRIVLVVLAASVALAGNNLNDQVNKYILPDRCRAGLQEQLNLELHAS LVYMQMAAHLANNKVARGGFARFFRDQSSEEREHAQKIIDYLNLRGGT VSAVNVDMPPTAIWMSVLDALQAALALEHRVTNRLYELHRLAEEYDAQ MADFLEQEFLAEQVRSIDQLQRLITQLQNMETGLGEFLLDQQLRA.

The signal sequence responsible for the protein secretion of the cell is marked in bold. The primary sequence enables correct folding of the protein to the quarter structure, which transports the non-heme iron from the tick gut to the peripheral tissues. In the absence of ferritin 2, or in the case of its successful blocking, the ticks are not able to finish feeding and dry directly on the host.

Example 4

Ferritin 2, designated as FER2-DV, is a protein produced by the tick Dermacentor variabilis and its primary structure is determined by the amino-acid sequence:

(SEQ ID. NO: 4) MLRLALLIALASAAWAGNNLNEQVNQNRYFLHDRCRIGLQEQVNLEL HASLVYMQMAAHLANNKVARNGFARFFRDQSSEEREHAQKLVDYVNL RGGTVSAVSVDMPATATWMSVLDALQAALALEHRVTNRLHELHRLAD DSQDPQMADFLEQEFLAEQVRSIDQLQRLITQLQNMDTGLGEFLLDQ QLRA.

The signal sequence responsible for the protein secretion of the cell is marked in bold. The primary sequence enables correct folding of the protein to the quarter structure, which transports the non-heme iron from the tick gut to the peripheral tissues. In the absence of ferritin 2, or in the case of its successful blocking, the ticks are not able to finish feeding and dry directly on the host.

INDUSTRIAL APPLICABILITY

Production of recombinant ferritin 2 and its use as an antigen for the host immunization against tick feeding and transmission of the tick-borne pathogens. 

1. Ferritin 2 for the host immunization against ticks which has the amino-acid sequence without the signal sequence in at least 85% identical to the amino-acid sequences of FER2-IR or FER2-IS or FER2-RM or FER2-DV and in the native conditions, it forms the quarter structure, which transports the non-heme iron from the tick gut to the peripheral tissues.
 2. Ferritin 2 for the host immunization against ticks according to the claim 1, which has the primary structure without signal sequence composed of amino-acid sequence of FER2-IR.
 3. Ferritin 2 for the host immunization against ticks according to the claim 1, which has the primary structure without signal sequence composed of amino-acid sequence of FER2-IS.
 4. Ferritin 2 for the host immunization against ticks according to the claim 1, which has the primary structure without signal sequence composed of amino-acid sequence of FER2-RM.
 5. Ferritin 2 for the host immunization against ticks according to the claim 1, which has the primary structure without signal sequence composed of amino-acid sequence of FER2-DV.
 6. A fragment, composed of the sequence region of ferritin 2, for the host immunization against ticks according to claim
 2. 